Fe-S proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and inorganic sulfur of various structures, termed Fe-S clusters. In addition to the well-established role of electron transport, Fe-S proteins are involved in a diverse range of non-redox processes including sensing and regulatory functions. In this application, we propose to employ a combined spectroscopic/rapid-kinetic approach to investigate the biosynthesis of Fe-S clusters and to study the newly discovered functional role of Fe-S cluster in stabilizing radical intermediates. It has been established that a pair of the nitrogen fixation gene products, NifU and NifS, are essential for the assembly of the Fe-S clusters for the nitrogenase enzyme system. Homologs of NifS and NifU, termed IscS and IscU, respectively, are found in a wide spectrum of living organisms ranging from bacteria to human, and thus, have been proposed to be involved in the general assembly/repair of Fe-S clusters in biology. Here, experiments are proposed to investigate the mechanism of Fe-S biosynthesis and to establish the roles play by NifU/NifS and IscU/IscS in this important biological process. For the purpose of enhancing our understanding of Fe-S cluster functions, three functionally diverse proteins were chosen for the proposed studies: pyruvate formate-lyase activating enzyme (PFL-AE), ferredoxin: thioredoxin reductase (FTR) and biotin synthase. PFL-AE activates pyruvate formate lyase (PFL) by catalyzing the generation of a glycyl radical in PFL. FTR catalyzes the reductive cleavage of disulfide groups in thioredoxins for enzyme activations, and biotin synthase converts dethiobiotin to biotin. Evidence accumulated so far suggests that all three enzymes employ a 4Fe-4S cluster-mediated site-specific u(3)-S(2-) based chemistry for their respective functions. The proposed study is designed to evaluate the validity of this suggestion and to determine the detailed mechanistic steps involved in the catalytic cycles. The methods of choice for the proposed studies are Mossbauer and EPR spectroscopies, which are particularly suited for the study of Fe-containing proteins. Rapid freeze-quench kinetic techniques will be used to trap reaction intermediates for spectroscopic characterization and for kinetic investigations. Whenever possible, other complementary techniques, such as resonance Raman, ENDOR, and EXAFS will be used to obtain further structural information on the reaction intermediates. Site-specific variants will be engineered, produced and subjected to similar kinetic/spectroscopic investigations for the purpose of defining the functional roles of specific residues. Detailed mechanistic insights at a molecular level are expected to emerge from the proposed investigations.